Bacteriophage phi6 contains three pieces of double-stranded RNA in its nucleocapsid. The pieces possess different genetic information but have similar termini. The packaging of the genomic segments is precise. The aim of this proposal is to determine how the packaging system is able to select one of each of the chromosomes. Specifically, the regions of the three chromosomes necessary for packaging will be identified. The order of packaging and the requirements for packaging in terms of amount of RNA will be determined in an in vitro packaging system that we have developed. The entire genome of phi6 has been cloned as cDNA and completely sequenced. Procapsids are produced from cDNA copies of segment L in both Escherichia coli and Pseudomonas phaseolicola, the natural host of phi6. These procapsids are capable of packaging single-stranded viral RNA, synthesizing the complementary minus strand to form double-stranded RNA and then transcribing this dsRNA to form plus ssRNA. Procapsids will be isolated and used to study both the replicase and packaging reactions. The composition of procapsids, will be varied so as to identify the proteins involved in general packaging, segment specific packaging, replication and transcription. RNA transcripts made from cDNA constructions of the genomic segments can be packaged by the procapsids. The regions of the RNA involved with packaging will be identified and manipulated so as to clarify the interaction between the RNA and the packaging and replication machinery. The nature of the mechanisms of selective packaging in viruses of segmented genomes is currently unknown. The elucidation of the mechanism for phi6 has relevance to the packaging of other segmented-genome viruses. Several members of the reoviridae, e.g., rotavirus and blue tongue virus, are the etiologic agents of important human and animal diseases.